The present invention relates to improved methods and bacterial strains for the production of nisin, in particular protein-engineered nisins.
Nisin is a highly modified peptide antibiotic produced, for example, by certain strains of Lactococcus lactis. It is of great interest to the food industry because of its efficient antimicrobial activity against a wide range of gram-positive organisms including many spoilage bacteria and food pathogens, for example, Listeria, Clostridia and Bacillus species (see Fowler and Gasson (1990) in Food Preservatives (eds N. J. Russell and G. W. Goulds) pages 135-152, Blackie and Sons, Glasgow, UK).
The chemical structure of nisin is well established (FIG. 1). It is a member of the family of antibiotics termed lantibiotics. These unusual polycyclic peptides share the structural features of dehydro-residues and intrachain sulphide bridges forming lanthionine and xcex2-methyllanthionine rings. The atypical residues are introduced by post-translational modification of amino acids serine, threonine and cysteine in the primary sequence of a precursor peptide (lantibiotics are the subject of a recent extensive review by Jung (1991) in Nisins and novel lantibiotics (eds Jury, G. and Sahl, H.-S.) pages 1-34, ESCOM, Leiden, Netherlands). Biosynthesis of nisin thus involves genes for both the inactive precursor of nisin, known as prenisin, (nisA) and also the modifying enzymes responsible for nisin maturation. The mature nisin molecule is based on a sequence of 34 amino acids. The protein encoded by nisA includes a 23 amino acid N terminal signal sequence which is cleaved off during secretion of nisin. The conversion of prenisin, encoded by nisA, into mature nisin involves cleavage of the leader and the modification of individual amino acids. A nisA gene has been cloned and characterised and shown to have a chromosomal location (see Dodd et al (1990) J. Gen. Microbiol. 136, 555-566). A number of additional genes involved in the enzymatic modification of prenisin, translocation and immunity are encoded by nisin producing strains (Kuipers et al (1993) Eur. J. Biochem. 216, 281-291; Engelke et al (1994) Appl. Environ. Microbiol. 60, 814-825).
Established protein engineering techniques can be used to introduce changes to the amino acid sequence of nisin. This involves modifying the coding region of the nisin structural gene, nisA, for example by site-directed or random mutagenesis. Expression of these changes is complicated by the fact that nisin is post-translationally modified.
Variant nisins may be constructed by the expression of variant nisA genes in a host strain which encodes the necessary maturation machinery, and thus can process the modified precursor peptide. One approach is to transform a nisin producing strain with a recombinant plasmid encoding variant nisA gene. In this background the host""s maturation enzymes are available to process both the resident prenisin and its plasmid-encoded variant. A strategy of this type has been reported for a strain that carries the wild-type nisin transposon (Kuipers et al (1991) in Nisins and novel lantibiotics (eds Jung, G. and Sahl, H.-S.), pages 250-259, ESCOM, Leiden, Netherlands). However, the disadvantage of this system is that both the host""s nisin and the engineered variant are synthesised together, making complex chemical separation procedures necessary prior to analysis of the properties of the novel peptide. Such a procedure would be particularly undesirable for industrial scale production of a variant nisin.
WO 93/20213 describes a process for producing a variant nisin from Lactococcus in the absence of natural nisin in which a plasmid-borne variant nisA gene (which encodes the variant nisin) is introduced into a strain of Lactococcus which does not secrete its natural nisA nisin (because the nisA gene has been inactivated) but is capable of expressing genes for nisin modification, immunity and translocation out of the cell.
WO 92/18633 discloses plasmid-based systems for the expression of variant nisins from the nisZ gene (or mutants thereof) in Lactococcus strains that do not produce natural nisA nisin.
Unexpectedly we have found that by replacing the natural, chromosomal copy of the nisA gene (or at least a part thereof) with a variant nisA gene (or part thereof) we can produce surprisingly high levels of nisin, particularly variant nisins, from Lactococcus. Thus, the present invention provides improved methods and organisms for producing variant nisins with greater efficiency.
One aspect of the invention provides a method for making a cell which does not contain a natural nisA gene but expresses a nisin comprising the step of providing a cell with a variant nisA gene and genes for nisin modification, secretion and immunity wherein the variant nisA gene has the same relationship as the natural nisA gene to the gene cluster containing the natural nisA gene and the genes for nisin modification, secretion and immunity.
A second aspect of the invention provides a cell which does not contain a natural nisA gene but expresses a nisin comprising a variant nisA gene wherein the variant nisA gene has the same relationship as the natural nisA gene to a gene cluster containing the natural nisA gene and the genes for nisin modification, secretion and immunity.
A third aspect of the invention provides a process for producing nisin comprising culturing a cell as described in the second aspect of the invention and obtaining the nisin produced thereby.
A fourth aspect of the invention provides a nisin produced by the process of the invention.
A fifth aspect of the invention provides the use of a nisin produced according to the process of the invention as an antimicrobial agent. The ability of nisin to inhibit growth of spoilage bacteria and food pathogens has resulted in the extensive use of nisin as a natural preservative in certain food products, particularly dairy products such as soft cheeses. Variant nisins are also used.